New specific and rapid assay for in situ apoptosis labeling for cancer studies Abstract In this project we will develop and validate in the context of commercial use a new express assay for molecular pathology of cancer and in situ studies. The assay will selectively label apoptotic, but not necrotic cells, based on detection of characteristic double-stranded DNA breaks produced by apoptotic executioner nucleases. At present these biomarkers are considered highly specific for programmed cell death, but their detection takes 24 hrs. The proposed technology will perform such specific detection within minutes, permitting cost-effective high throughput in situ assessments of anticancer drugs and therapies. The assay will use the unique properties of viral DNA topoisomerase, we synthesized de novo, starting from its sequence in the genome of the giant Acanthamoeba polyphaga virus. This archaic enzyme sequence was acquired by the virus at some point in its evolution from a now extinct animal species. We found that this recombinant protein possesses extremely fast ligation activity. The topoisomerase can rapidly ligate DNA ends, finishing the reaction within several seconds after addition to DNA. This surpasses the speed of all known DNA ligases by two orders of magnitude. It permits a novel ultra-fast topoligation labeling of apoptotic cells in tissue sections. This new assay will have high commercial potential and will offer advantages of speed and specificity over the currently available conventional in situ approaches. It will be particularly useful in research, where large-volume quantitations of programmed cell death cells are essential, such as in anti- cancer pro-apoptotic drug development and in molecular pathology studies of tumors. The Specific Aims of the proposal are: 1) To develop and validate in the context of commercial use the first express assay for specific detection of apoptosis in histological sections. The assay will operate by the extra-fast topoligation labeling of blunt-ended DNA breaks with terminal 5?PO4 produced by apoptotic executioner nucleases. To verify the new labeling approach using in vitro models, and to ensure its reproducible and robust labeling, essential for the ease of use and the future competitive advantage. 2) To validate the new express assay in the context of commercial use by using apoptotic models related to cancer. To optimize it for the ease of use by potential customers by ensuring high and consistent speed of detection, sensitivity, and specificity. Verify its general applicability in different samples including fixed cells and tissue sections. Ensure high reproducibility of labeling results and high signal-to-noise ratio, providing strong and unobscured labeling of specific DNA breaks in individual apoptotic cells in different cancer samples.